1.4. Immune signatures in chronic inflammatory diseases: focus on Interleukin-6

One of the key players in chronic inflammation and inflamm-aging is Interleukin 6 (IL-6). Dysregulation of the IL-6 axis plays a central role in GCA, RA and type 2 diabetes. Interleukin 6 (IL-6) is a pleiotropic cytokine involved in many biologic functions that affect the immune system, the vasculature and organs. In general, classic IL-6 signaling via the transmembrane IL-6 receptor (IL- 6R) is thought to be responsible for the anti-inflammatory properties of IL-6, whereas trans-signaling via the trans-signaling pathway upon binding to the soluble form of IL-6R (sIL-6R) is responsible for the proinflammatory actions of IL-6. It is generally accepted that inflamm-ageing of both the immune system and the vascular wall of large vessels are triggers for disease development in GCA and possibly also in type 2 diabetes. Vascular dendritic cells and CD4+ T cells play an important role in the initiation of vessel inflammation and in granuloma formation. Moreover, two dominant pro-inflammatory cytokine clusters have been linked to disease activity in GCA: the Interleukin(IL)-12-IFNγ axis promoting T helper (h)1 responses and the IL-6-IL-17 axis promoting Th17 responses. Recently, data from the GiACTA trial demonstrated that Tocilizumab, a blocker of the IL-6 Receptor (IL-6R; CD126), is effective at achieving sustained, glucocorticoid-free remission in GCA patients. The cellular mechanisms, however, by which IL-6 drives the disease remain to be resolved.

The overarching hypothesis of the current proposal is that dysregulation of interleukin 6 (IL-6) axis is a common mechanism responsible for the individual variety in disease course and treatment response in GCA, RA and type 2 diabetes. The current proposal aims to establish the consequences of chronic IL-6 exposure on survival, expansion and commitment of especially naïve CD4+ T cells towards autoimmune effector cells in the development of GCA. In addition, the consequences of circulating (physiological) levels of IL-6 on regulatory T cells will be assessed in GCA, RA and type 2 diabetes.

In order to explore the modulating role of the IL-6 axis on CD4+ T cell subsets we will investigate total CD4 and CD4 subsets, examine expression of IL-6R and gp130 on CD4+ T cells and subsets and assess IL-6, soluble IL-6R and soluble gp130 (inhibitor of IL-6 signaling) in sera of the participating cohorts. We will assess the effect of both the classical IL-6R signaling and IL-6 trans signaling pathways (IL-6+sIL-6R) on naïve T cell activation, proliferation and Th1/Th17 skewing potential and on Treg dysregulation. Bio repositories and patient cohorts

GCA, healthy control and RA cohort

Unique cohorts of newly-diagnosed, untreated GCA patients have been built at the UMCG Vasculitis Expertise Centre. From these patients serial blood samples/ peripheral blood mononuclear cells PBMCs at the time of diagnosis and at regular intervals at follow up have been prospectively collected and bio-banked (total cohort contains more than 100 patients and inclusion and follow up is ongoing) . Treatment is done according to a fixed protocol including tocilizumab which has recently been registered for GCA. Our SENEX cohort including around 100 aged individuals serves as a healthy, age-matched control group. For RA we have a unique follow up cohort of patients at risk for RA defined as autoantibody ( ACPA+ and/or RF+) positive patients without arthritis. During follow up 40% of RA risk patients developed RA. We also have stored PBMC samples from seropositive RA (ACPA+ and/or RF+) patients and recently diagnosed seronegative ( ACPA- and RF-) RA patients (more than 150 patients in total).

Diabetes cohort

In the second part of this project the discovered inflammatory signatures will be measured in patients with type 2 diabetes. We manage a large observational study of patients with type 2 diabetes in primary care in Groningen. A total of 903 patients are participating in this study. Blood and urine samples are collected at baseline and at annual visits. In a sub group of patients also PBMCs will be collected. The follow-up duration ranges from 3 to 6 years. This cohort provides a unique opportunity to translate the inflammation signatures from GCA and RA cohorts to diabetes and to test to what extend they are common in both chronic conditions.

1. Evidence for elevated IL-6 signaling in CD4+ T cell differentiation subsets (with emphasis on naïve CD4+ T cells) in GCA/PMR?
2. What is the contribution of IL-6 to naïve CD4 T cell survival, expansion and differentiation?
3. What is the effect of chronic IL-6 stimulation on Treg functionality?
4. Can CD4+ T cell IL-6 signatures help stratify GCA patients for personalized medicine?
5. Is there a differential treatment response between GCA patients with high IL-6 levels versus those with low levels?
6. Do CD4+ T cell IL-6 signatures translate to type 2 diabetes and can then be used to select subgroup of patients with a pro-inflammatory phenotype more likely to develop atherosclerotic / vascular disease?

A better understanding of the factors governing CD4 T cell driven autoimmune diseases is highly awaited as it is likely to provide a rational for novel targets of treatment. Interleukin 6 (IL-6) being a pleiotropic cytokine is hypothesized to be responsible for the individual variety in early disease course and early treatment response in GCA, RA and type 2 diabetes. Insight into the individual response within the different disease backgrounds will contribute to identifying serum biomarkers reflecting the immune signatures in these chronic inflammatory diseases which will ultimately form the foundation for stratification of patients for personalized medicine.

• Biologicals targeting immune subsets and chemical drugs targeting signaling pathways can be designed based on insights into common immune signatures in chronic inflammatory diseases for prediction of disease progression.
• Also especially in RA there is a wealth of drugs targeting specific pathways which can be used and translated to GCA and type 2 diabetes
• Lastly most drugs still come at a price of immune suppression and subsequent vulnerability to infectious diseases. Creating an urgent need for precision medicine.
• The project links to various other research projects within ProminenT. Bischoff investigates chronic inflammatory phenotypes in obese and diabetic individuals and aims to develop new methodologies to measure inflammatory metabolites. We will discuss with Bischoff if we can use his methodology in our cohorts as well. Poelarends will probably focus on inflammatory drug development and it may be of interest to discuss if the drugs that he will synthesize affect IL-6 pathways and apply to GCA/RA/Diabetes cohorts as well.